Development of cell-penetrating peptides targeting nuclear and extra-nuclear signaling pathways in cancer cells

Abstract

Growth factors and hormones often mediate their biological effects through series of protein-protein interaction between cytoplasmic signaling molecules or transcription factors. Interfering of these protein-protein interactions has been shown to reduce or inhibit biological and growth responses to various growth factors and hormones. We previously demonstrated that progesterone receptor (PR) contains a polyproline domain (PPD) which directly interact to Src homology 3 (SH3) domain-containing molecules and expression of PR-PPD inhibits EGFR-mediated NSCLC cell proliferation. In this study, we investigated that the introduction of PR-PPD by cell-penetrating peptide could inhibit EGF-induced NSCLC cell proliferation. PR-PPD was attached to a cancer-specific CPP, Buforin2 (BR2), to help deliver the PR-PPD into NSCLC cells. Addition of BR2-2xPPD peptide containing two PR-PPD repeats was more effective in inhibiting NSCLC proliferation and significantly reduced EGF-induced phosphorylation of Erk1/2. BR2-2xPPD treatment could induce cell cycle arrest by inhibiting the expression of cyclin D1 and CDK2 gene in EGFR-wild type A549 cells. The combination treatment of EGFR-TKIs with BR2-2xPPD peptide was more effectively suppressed growth of NSCLC PC9 cells harboring EGFR mutation as compared to EGFR-TKIs treatment alone. Additionally, BR2-2xPPD peptide could mediate growth inhibition in acquired gefitinib- and erlotinib- resistance lung adenocarcinoma cells. In addition to PR-PPD, previous studies had demonstrated that peptide containing LXXLL motifs inhibited interactions between steroid hormone receptor and its coactivator. In this study, BR2-LXXLL peptide was designed from GRIP-1 protein which had high potential to inhibit ER transcription. The nuclear localization of SV40 was also added into peptide in order to facilitate access to nucleus. Our results demonstrated that BR2-LXXLL peptide could effectively inhibit the transcriptional activity of PR in T47DC42 breast cancer cell expressing PR-B isoform while the scramble peptide abolished this inhibition. BR2-LXXLL and BR2-2xPPD treatments dose-dependently decreased Estrogen-induced cell proliferation in ER-positive MCF-7 breast cancer cell lines. Moreover, BR2-LXXLL and BR2-2xPPD peptides also significantly reduced triple-negative breast cancer cell growth which lack of targeted therapy. Altogether, our data suggested that the cancer cell specific peptide of PR-PPD and LXXLL motifs could be used to further develop as novel anticancer treatment in the near future.